Efficient persistence of extrachromosomal KSHV DNA mediated by latency- associated nuclear antigen

Science

Volumn 284, Issue 5414, 1999, Pages 641-644

  Ballestas, Mary E ^a   Chatis, Pamela A ^b,c   Kaye, Kenneth M ^a  

^a BRIGHAM AND WOMEN S HOSPITAL   (United States)

^b HARVARD MEDICAL SCHOOL   (United States)

^c NEN Life Science Products   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CELL NUCLEUS ANTIGEN; DNA FRAGMENT; VIRUS ANTIGEN; VIRUS DNA;

ANTIGEN EXPRESSION; ARTICLE; CELL NUCLEUS; CELLULAR DISTRIBUTION; EPISOME; GENE SEGREGATION; HERPES VIRUS; INTERPHASE; KAPOSI SARCOMA; LYMPHOBLAST; MITOSIS; PERSISTENT VIRUS INFECTION; PRIORITY JOURNAL; PROGENY;

ANTIGENS, VIRAL; CELL NUCLEUS; CHROMOSOMES; COSMIDS; DNA, VIRAL; HERPESVIRUS 8, HUMAN; HUMANS; INTERPHASE; LYMPHOCYTES; MICROSCOPY, CONFOCAL; MITOSIS; NUCLEAR PROTEINS; PLASMIDS; TRANSFECTION; TUMOR CELLS, CULTURED;

HERPES; HERPESVIRIDAE; HUMAN HERPESVIRUS 8; LANA;

EID: 0033597453     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5414.641     Document Type: Article

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27. 2-terminal LANA amino acids are replaced with the FLAG epitope (DYKDDDDKV) (22). LANA stable cell lines were generated by electroporating pSG5LANA or pSG5 F-LANA and a plasmid encoding the hygromycin-resistance gene downstream of an SV40 promoter into BJAB cells [K. M. Kaye, K. M. Izumi, G. Mosialos, E. Kieff, J. Virol. 69, 675 (1995); D. Liebowitz, J. Mannick, K. Takada, E. Kieff, ibid. 66, 4612 (1992)]. After 48 hours, cells were seeded into microtiter plates, and hygromycin-resistant clones were selected. Hygromycin-resistant BJAB cells expressing pSG5LANA or pSG5 F-LANA were transfected with 25 μg of the Z6 or Z8 cosmid. Z6 contains KSHV sequence including the terminal repeats and the first ∼33 kb of the BC-1 genome cloned into S-Cos1 (Stratagene), and Z8 contains nucleotides ∼73,000 to 107,000 of BC-1 KSHV cloned into S-Cos1 (8). S-Cos1 encodes the neomycin-resistance gene downstream of an SV40 promoter and provides G418 resistance. After 48 hours, cells were placed under G418 selection. For subclone analysis, the Z6 cosmid was digested with Hind III and religated, resulting in the deletion of KSHV sequences after the first ∼13 kb of the genome (Z6-13). The two largest remaining Z6 KSHV Hind III fragments of ∼7 and ∼11 kb were cloned into pREP9 (Invitrogen) after the deletion of the pREP9 sequences between Cla I and Kpn I (Z6-7 and Z6-11 respectively). pREP9 encodes the neomycin-resistance gene downstream of a thymidine kinase promoter and provides G418 resistance. The Z6 subclones were transfected into BJAB/F-LANA or BJAB cells and selected for G418 resistance as above.
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